Stabilization of implantable bioprosthetic tissue

ABSTRACT

The invention relates to implantable bioprostheses (e.g. implantable biological tissues) and to compositions and methods for stabilizing them. Implantable bioprostheses stabilized as described herein exhibit improved mechanical properties and reduced post-implantation calcification. The implantable bioprosthesis is made by contacting a bioprosthesis (e.g. a tissue obtained from an animal or an article comprising a tissue and a synthetic material) with a polyepoxy amine compound.

FIELD OF THE INVENTION

[0001] The field of the invention is stabilization of implantablebioprosthetic devices and tissues.

BACKGROUND OF THE INVENTION

[0002] Surgical implantation of prostheses and tissues derived frombiological sources, collectively referred to herein as bioprostheticdevices or bioprostheses, is an established practice in many fields ofmedicine. Common bioprosthetic devices include heart valves, pericardialgrafts, cartilage grafts and implants, ligament and tendon prostheses,vascular grafts, skin grafts, dura mater grafts, and urinary bladderprostheses. In the case of valvular prosthetic devices, bioprosthesesmay be more blood compatible than non-biological prostheses because theydo not require anticoagulation therapy.

[0003] Bioprosthetic devices include prostheses which are constructedentirely of animal tissue, and combinations of animal tissue andsynthetic materials. Furthermore, a biological tissue used in abioprosthetic device can be obtained or derived from the recipient(autogeneic), from an animal of the same species as the recipient(allogeneic), from an animal of a different species (xenogeneic), oralternatively, from artificially cultured tissues or cells. Irrespectiveof the source of the tissue, major objectives in designing abioprosthetic device include enhancement of durability and reduction ofbiomechanical deterioration in order to enhance the functional enduranceof the device.

[0004] The material stability of bioprosthetic devices can becompromised by any of several processes in a recipient, including, forexample, immune rejection of the tissue, mechanical stress, andcalcification. Implantation of biological tissue that is not pre-treated(i.e. stabilized prior to implantation) or is implanted without priorsuppression of the recipient's immune system can induce an immuneresponse in the recipient directed against the tissue. Identification ofbioprosthetic tissue as ‘non-self’ by the immune system can lead todestruction and failure of the implant. Even in the absence of an immuneresponse, mechanical stresses on implanted tissue can induce changes inthe structure of the bioprosthesis and loss of characteristics importantto its mechanical function. In addition to these degradative processes,calcification of bioprosthetic tissue (i.e. deposition of calcium andother mineral salts in, on, or around the prosthesis) can substantiallydecrease resiliency and flexibility in the tissue, and can lead tobiomechanical dysfunction or failure. In order to extend the useful lifeof bioprosthetic devices by improving their mechanical properties andmitigating their antigenic properties, the devices can be treated priorto implantation using a variety of agents. These pre-treatment methodsare collectively referred to in the art as fixation, cross-linking, andstabilization.

[0005] Glutaraldehyde is the most common stabilizing reagent used fortreatment of valvular and other collagen-rich bioprosthetic devices.Glutaraldehyde is a cross-linking agent which has been used forpre-implantation stabilization of tissues, both alone and in combinationwith a variety of other reagents including diisocyanates, polyepoxideethers, and carbodiimides. Pre-treatment using glutaraldehyde and,optionally, other reagents, stabilizes implantable tissue with respectto both immune reactivity and mechanical stress by covalently linkingproteins and other structures on and within the tissue. Cross-linking ofa bioprosthetic tissue can be accompanied by treatment with anadditional reagent (e.g. ethanol) to retard post-implantationcalcification of the tissue. Use of glutaraldehyde as a stabilizingreagent can accelerate prosthesis calcification and necessitates use ofa calcification inhibitor. Known calcification inhibitors includeethanol, aluminum chloride, chondroitin sulfate, andaminopropanehydroxyphosphonate (APD).

[0006] A significant need exists for compositions and methods capable ofstabilizing bioprosthetic devices and reducing post-implantationcalcification. The present invention provides such compositions andmethods.

BRIEF SUMMARY OF THE INVENTION

[0007] The invention relates to an implantable bioprosthesis comprisingproteins cross-linked with a poly-(2-hydroxyorgano)amino moiety. Thebioprosthesis can be substituted with (i.e. reacted with a polyepoxyamine compound to yield) the poly-(2-hydroxyorgano)amino moiety at twoor more epoxy-reactive moieties of the bioprosthesis, such as amethylthio group, a primary amine group, a phenolic hydroxyl group, aphosphate group, or a carboxyl group. For example, substantially allepoxy-reactive groups at the surface of the bioprosthesis can besubstituted with (i.e. reacted such that they are linked by)poly-(2-hydroxyorgano)amino moieties. The bioprosthesis can, forexample, be any one of an artificial heart, a heart valve prosthesis, anannuloplasty ring, a dermal graft, a vascular graft, a vascular stent, astructural stent, a vascular shunt, a cardiovascular shunt, a dura matergraft, a cartilage graft, a cartilage implant, a pericardium graft, aligament prosthesis, a tendon prosthesis, a urinary bladder prosthesis,a pledget, a suture, a permanently in-dwelling percutaneous device, asurgical patch, a coated stent, and a coated catheter. Thepoly-(2-hydroxyorgano)amino moiety can, for example, be apoly-(2-hydroxypropyl)amino moiety, such as that formed by reactingtriglycidyl amine with epoxy-reactive groups of the bioprosthesis.

[0008] The implantable bioprosthesis can be one which comprises abiological tissue (e.g. a heart, a heart valve, an aortic root, anaortic wall, an aortic leaflet, a pericardial tissue, a connectivetissue, dura mater, a bypass graft, a tendon, a ligament, a dermaltissue, a blood vessel, an umbilical tissue, a bone tissue, a fascia, ora submucosal tissue). Such a tissue can be harvested from an animal(e.g. a human, a cow, a pig, a dog, a seal, or a kangaroo).Alternatively, the implantable bioprosthesis can be one which comprisesa synthetic analog of a bioprosthetic tissue.

[0009] The proteins of the bioprosthesis can be cross-linked bycontacting the bioprosthesis with an polyepoxy amine compound, forexample in an aqueous liquid having a pH of about 6 to 10, about 7 to10, or about 7.0 to 7.4. An exemplary polyepoxy amine compound istriglycidyl amine.

[0010] The implantable bioprosthesis can be treated with a secondstabilization reagent in addition to the polyepoxy amine compound. Forexample, the second stabilization reagent can be aglycosaminoglycan-stabilizing reagent (e.g. a carbodiimide), across-linking reagent, or a calcification inhibitor (e.g. aluminumchloride).

[0011] The invention also includes an implantable bioprosthesis made bycontacting an implantable bioprosthesis and a polyepoxy amine compound.The bioprosthesis is thereby stabilized.

[0012] In addition, the invention includes a method of stabilizing animplantable bioprosthesis. The method comprises contacting thebioprosthesis and a polyepoxy amine compound in order to stabilize thebioprosthesis.

[0013] In another aspect, the invention relates to a composition forstabilizing an implantable bioprosthesis. This composition comprises apolyepoxy amine compound and at least one of a calcification inhibitor,a glycosaminoglycan-stabilizing reagent, and a second cross-linkingreagent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 depicts a synthetic method for preparing triglycidyl amine(TGA).

[0015]FIG. 2 is the ¹H NMR spectrum of TGA synthesized as describedherein.

[0016]FIG. 3 is a graph which indicates the temporal change in the pH ofthe cross-linking solution in which porcine heart valve cusps weretreated using two different stabilization methods described herein.

[0017]FIG. 4 is a graph which indicates the temporal change in thethermal shrinkage temperature of porcine heart valve cusps treated usingtwo stabilization methods described herein, as assessed usingdifferential scanning calorimetry.

[0018]FIG. 5 is a graph which indicates the temporal change in thethermal shrinkage temperature of porcine heart valve cusps treated usingthree stabilization methods described herein, as assessed usingdifferential scanning calorimetry.

[0019]FIG. 6 is a diagram which depicts reaction of a generalized epoxyamine compound with a reactive moiety designated ‘Z’ in the figure toyield a 2-hydroxyorganoamine moiety bound with the Z moiety.

[0020]FIG. 7 is a diagram which depicts the initial reaction productformed by reaction of triglycidyl amine with a reactive moietydesignated ‘Z’ in the figure to yield a2-hydroxy3-(diglycidylamino)propyl moiety bound with the Z moiety.

[0021]FIG. 8 is a diagram which depicts reaction of a generalized epoxyamine compound with a methylthio moiety of a large molecule (e.g. amethionine residue side chain in a protein). The large molecule isrepresented by a wavy line. X is a non-reactive, biocompatible anion.

[0022]FIG. 9 is a diagram which depicts reaction of a generalized epoxyamine compound with a primary amine moiety of a large molecule (e.g. alysine residue side chain in a protein). The large molecule isrepresented by a wavy line. X is a non-reactive, biocompatible anion. Asshown in the figure, reaction of the alkylamino moiety with the epoxyamine compound can result in addition of one, two, or three2-hydroxyalkylamino moieties thereto.

[0023]FIG. 10 is a diagram which depicts reaction of a generalized epoxyamine compound with a phenolic hydroxyl moiety of a large molecule (e.g.a tyrosine residue side chain in a protein). The large molecule isrepresented by a wavy line.

[0024]FIG. 11 is a diagram which depicts reaction of a generalized epoxyamine compound with a phosphate moiety of a large molecule (e.g. amonoalkyl phosphate, such as a phosphatidyl serine residue side chain ina protein). The large molecule is represented by a wavy line.

[0025]FIG. 12 is a diagram which depicts reaction of a generalized epoxyamine compound with a carboxyl moiety of a large molecule (e.g. aglutamate residue side chain in a protein). The large molecule isrepresented by a wavy line.

[0026]FIG. 13 is a diagram which depicts polymerization of triglycidylamine in solution. X is a non-reactive, biocompatible anion.

DETAILED DESCRIPTION

[0027] The invention relates to stabilized implantable bioprostheses andto a method of stabilizing an implantable bioprosthesis (e.g. animplantable biological tissue or synthetic tissue-containing ortissue-like implant) using one or more of a class of compounds notpreviously used for this purpose. The stabilization method involvescontacting the bioprosthesis with a polyepoxy amine compound. Thebioprosthesis can also be treated with one or more additional reagentsin order to further stabilize it, as described herein.

[0028] Definitions

[0029] As used herein, each of the following terms has the meaningassociated with it in this section.

[0030] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0031] A “bioprosthesis” is an implantable protein-containing article,all or part of which comprises a biological tissue (e.g. a tissueobtained from an animal or a cultured animal tissue, such as tissueobtained from a human or a cultured human tissue), a component of such atissue (e.g. cells or extracellular matrix of the tissue), or somecombination of these. Bioprostheses specifically retain and enablebiologic structure and function in their intended implant configuration.Examples of bioprostheses or components include, but are not limited to,an artificial heart, a heart valve prosthesis, an annuloplasty ring, adermal graft, a vascular graft, a vascular, cardiovascular, orstructural stent, a vascular or cardiovascular shunt, a dura matergraft, a cartilage graft, a cartilage implant, a pericardium graft, aligament prosthesis, a tendon prosthesis, a urinary bladder prosthesis,a pledget, a suture, a permanently in-dwelling percutaneous device, anartificial joint, an artificial limb, a bionic construct (i.e. one ofthese bioprostheses comprising a microprocessor or other electroniccomponent), and a surgical patch.

[0032] “Implantation,” and grammatical forms thereof, refers to theprocess of contacting a prosthesis (e.g. a bioprosthesis) with a tissueof an animal in vivo wherein the contact is intended to continue for aperiod of hours, days, weeks, months, or years without substantialdegradation of the prosthesis. Such contact includes, for example,grafting or adhering the prosthesis to or within a tissue of the animaland depositing the prosthesis within an orifice, cavity, incision, orother natural or artificially-created void in the body of the animal.

[0033] “Stabilization,” and grammatical forms thereof, of abioprosthesis means increasing the mechanical strength of thebioprosthesis, decreasing the rate or incidence of degradation of thebioprosthesis following its implantation in or on an animal, or somecombination of these. Causes of degradation include mechanical wear,reactions between the prosthesis and the animal's immune system, andcalcification associated with the prosthesis. Stabilization can enhanceone or more of the durability, shelf life, and fatigue life of thebioprosthesis. Exemplary means of stabilizing a bioprosthesis includecovalently linking (“cross-linking”) components (e.g. proteins) of theprosthesis, inhibiting calcification associated with the prosthesis,co-incorporating a beneficial polymer or another agent into thebioprosthesis, and stabilizing a glycosaminoglycan (GAG) on theprosthesis or a tissue associated with the prosthesis. For example, aGAG is “stabilized” on a tissue when the tissue is reacted with areagent which generates at least two covalent bonds associated with aGAG molecule. These bonds can either be intramolecular or intermolecularin nature. A reagent which generates such bonds is herein designated a“GAG-stabilizing reagent.” The terms “stabilization,” “fixation,” and“cross-linking” are used interchangeably herein.

[0034] A GAG or protein is “endogenous” with respect to a tissue if theGAG or protein is normally present on or in the tissue in a healthyindividual which naturally comprises the tissue (e.g. a GAG or proteinwhich naturally occurs on or in the tissue, regardless of whether theGAG or protein was isolated with the tissue or was added to the tissueafter isolation thereof). Otherwise, the GAG or protein is “exogenous”with respect to the tissue.

[0035] A “polyepoxy amine compound” is a chemical species containingboth an amine moiety (e.g. a primary, secondary, tertiary, or quaternaryamine moiety, such as an oligomer of triglycidyl amine) and a pluralityof epoxide moieties. Polyepoxy amines in this group include, forexample, diepoxy amines and triepoxy amines.

[0036] A “poly-(2-hydroxyorgano)amino moiety” is a moiety formed byreaction of a polyepoxy amine compound with a plurality ofepoxy-reactive moieties of one or more substrate molecules (e.g. aprotein or a bioprosthesis).

[0037] “-Organo-”, in the context of a poly-(2-hydroxyorgano)aminocompound, refers to a carbon-containing moiety (e.g. an alkyl group suchas a C₁-C₆ straight chain alkyl group) interposed between an epoxy aminemoiety and an amine moiety of the compound.

[0038] An “epoxy-reactive moiety” is a moiety capable of reacting withan epoxide ring such that the epoxide ring is opened and a covalent bondis formed between the moiety and an atom of the epoxide ring.

[0039] Description

[0040] The invention relates to stabilization of a bioprosthesis, suchas an implantable biological tissue or an implant comprising aprotein-containing matrix. This stabilization is effected by contactingthe bioprosthesis with a polyepoxy amine compound, preferably in thepresence of an aqueous liquid, to yield an implantable bioprosthesiscomprising proteins cross-linked with one or morepoly-(2-hydroxyorgano)amino moieties. The polyepoxy amine can formcovalent bonds between chemical groups on or within the bioprosthesis,including amine groups (e.g. primary, secondary, and tertiary aminegroups), thio groups (e.g. thiol and methylthio groups), hydroxyl groups(e.g. phenolic and carboxylic hydroxyl groups of tyrosine, aspartate,glutamate side chains in a protein) and phosphate groups. As a result ofcontacting the bioprosthesis with the polyepoxy amine compound, covalentchemical bonds are formed between the polyepoxy amine compound and thebioprosthesis, such as bonds between a polyepoxy amine compound and oneor more amino acid residue side chains (e.g. side chains of one or moreproteins, including both proteins which are endogenous to a tissue ofthe bioprosthesis and exogenous proteins), between one molecule of apolyepoxy amine compound and another molecule of the same polyepoxyamine compound, or between both an amino acid residue side chain andanother molecule of the polyepoxy amine compound. Use of a polyepoxyamine compound results in formation of covalent linkages betweenepoxy-reactive moieties of the bioprosthesis. Thus, a network ofinter-connected chemical group's of the bioprosthesis (e.g.interconnected amino acid residue side chains) is generated, therebystabilizing the bioprosthesis. If the bioprosthesis is contacted with apolyepoxy amine compound in the presence of another compound (e.g. aprotein which does not normally occur in the bioprosthesis), then theother compound can be linked to the bioprosthesis.

[0041] Examples of the chemical moieties formed upon reaction of anepoxy amine compound with a chemical moiety of a bioprosthesis areillustrated in FIGS. 6-12. Of course, a plurality of such moieties canbe formed upon reaction of a polyepoxy amine compound with moieties of abioprosthesis, the plurality of moieties being linked by the moietydesignated “organoamino” in FIGS. 6 and 8-12 (i.e. linked by apoly-(2-hydroxyorgano)amino moiety, the designation ‘organoamino’referring to the portion of the poly-(2-hydroxyorgano)amino moiety otherthan the hydroxyethyl portion depicted in the formulas in each of FIGS.6 and 8-12).

[0042] In FIG. 6, reaction of an epoxy amine or polyepoxy amine compoundwith an epoxy-reactive moiety (“Z”) of a bioprosthesis leads to openingof the epoxide ring and formation of a covalent bond between one of thecarbon atoms of the epoxide ring and the Z moiety, thereby yielding theZ moiety having a 2-hydroxyorganoamine moiety attached thereto. Forexample, when the epoxy amine compound is triglycidyl amine (TGA, atriepoxy amine), reaction of TGA with a Z moiety of a bioprosthesisleads to formation of a covalent bond between the Z moiety and a2-hydroxy-3-(diglycidylamino)propyl moiety, as shown in FIG. 7. Thenon-reacted epoxide moieties of the 2-hydroxy-3-(diglycidylamino)propylmoiety can react with one or two other epoxy-reactive moieties of themolecule having the Z moiety, or they can react with one or twoepoxy-reactive moieties of other molecules to covalently link themoieties of the molecule or molecules. Similarly, a polyepoxide aminecompound can react with a number of epoxy-reactive moieties equal to thenumber of epoxide rings in the compound. A molecule or article (e.g. aprotein, a bioprosthesis, or a tissue) having a plurality ofepoxy-reactive moieties with which a polyepoxy amine compound (includinga polyepoxy amine compound such as TGA) has reacted is said to be“substituted with a poly-(2-hydroxyorgano)amino moiety”. The 2-hydroxygroup can further react with an epoxide moiety such that cross-linkingnetworks can be formed. The ether linkages formed from the 2-hydroxygroups are considered 2-hydroxy moieties in the claims below.

[0043] The reactive moiety with which an epoxy amine compound reactscan, for example, be a methylthio moiety (e.g. the methylthio moiety ofa methionine residue in a protein) as depicted in FIG. 8, a primaryamine moiety (e.g. the primary amine moiety of a lysine residue in aprotein) as depicted in FIG. 9, a phenolic hydroxyl moiety (e.g. thehydroxyl moiety of a tyrosine residue in a protein) as depicted in FIG.10, a phosphate moiety (e.g. the phosphate moiety of a phosphatidylserine residue in a protein) as depicted in FIG. 11, or a carboxylmoiety (e.g. the carboxyl residue of a glutamate residue in a protein)as depicted in FIG. 12. In FIGS. 8 and 9, “X” is preferably anon-reactive, biocompatible anion such as chloride or acetate.

[0044] The invention includes stabilized, implantable bioprostheseswhich have a plurality of epoxy-reactive moieties which have beenreacted with a polyepoxy amine compound (i.e. implantable bioprostheseshaving epoxy-reactive moieties which are cross-linked with one or morepoly-(2-hydroxyorgano)amino moieties). For example, all, substantiallyall, or a fraction (e.g. 90%, 80%, 70%, 50%, 25%, 10%, 5%, or 1% orfewer) of surface epoxy-reactive moieties of the bioprosthesis can becross-linked with one or more poly-(2-hydroxyorgano)amino moieties.

[0045] The polyepoxy amine compound has at least two epoxide moieties,and can have three or more. Preferably, the polyepoxy amine compound isone, such as TGA, in which the epoxide ring is separated from thenearest amino moiety by from 1 to 5 other atoms (e.g. a C₁-C₅ branchedor linear alkylene chain such as the methylene group which separates theepoxide ring and the tertiary amine moiety in TGA). Other chemicalgroups which can be interposed between the epoxide ring and the nearestamino moiety include, for example, branched or linear alkenyl chains.

[0046] In another embodiment, the polyepoxy amine compound is a polymerhaving a plurality of epoxide groups attached thereto (e.g. at one orboth ends or as side chains within or throughout the polymer). It isrecognized that some polyepoxy amine compounds have an epoxy-reactivemoiety (e.g. a non-quaternary amine moiety) and can undergoautopolymerization, giving rise to linear or branched polymers. Thelength or degree of branching can be controlled by, for example,modulating the length of time the polyepoxy amine preparation ispermitted to autopolymerize. For example, TGA is a tertiary amine whichcan autopolymerize, as shown in FIG. 13. When such a polymer is used,the polymer can be formed by polymerizing polyepoxy amine compoundmolecules with a polyepoxy amine compound molecule that is already boundwith one or more moieties of the bioprosthesis, by polymerizing thepolyepoxy amine compound prior to contacting it with the bioprosthesis,or both. When a polyepoxy amine polymer is used, the polymer can be alinear polymer or a branched polymer, and preferably has a molecularweight of about 185 to 10,000. For example, a polymer of TGA can be usedin which the polymer is formed by polymerization of at least about 15TGA molecules, yielding a TGA polymer having a molecular weight greaterthan 3000. As indicated in FIG. 13, polymerization of a polyepoxy aminecompound can lead to formation of a polymer having a plurality ofquaternary ammonium moieties. Prior art polyepoxide compounds (e.g.Denacol™ products) do not contain amino groups, and thus do notauto-polymerize to form polyepoxy amine compound polymers havingquaternary ammonium groups. While not being bound by any particulartheory of operation, it is believed that the quaternary ammoniummoieties in the polyepoxy amine compound polymers described herein are,at least in part, responsible for the improved stabilization propertiesof bioprostheses treated with such polymers.

[0047] Polyepoxy amine compounds can be prepared using synthetic methodsknown in the art (.e.g. by a modification of Ross et al., 1963 J. Org.Chem. 29:824-826 described herein, or as described in Martyanova et al.,1990, Sb. Nauch. Tr. Lenengr. In-t Kinoinzh. 2:139-141 {Chem. Abst. nos.116:43416 and 116:31137} or Chezlov et al., 1990, Zh. Prikl. Khim.(Leningrad) 63:1877-1878 {Chem. Abst. No. 114:121880}). Prior artapplications of these compounds have been limited to their use inindustrial resins and photoemulsions. It is believed that the presentdisclosure represents the first description of using polyepoxy aminecompound in a biomedical application.

[0048] The polyepoxy amine compound can be used at substantially anyconcentration. However, it is preferred that the concentration of thepolyepoxy amine compound be high enough to yield an appreciable rate ofreaction, but not so high that significant (e.g. greater than 50%)cytotoxicity occurs. For example, the rate of reaction can be such thata degree of cross-linking not less than about 50% that achievable usingglutaraldehyde (i.e. as assessed, for example by determining the thermalshrinkage temperature using, e.g., differential scanning calorimetry) isattained within not more than about 30 days, and preferably within notmore than 10 days of reaction. By way of example, when the polyepoxyamine compound is triglycidyl amine, a preferred range of concentrationsis about 0.01 to 1 molar, and is preferably about 100 millimolar for thereaction conditions described herein.

[0049] The aqueous liquid used in the stabilization method should,especially for tissue-containing bioprostheses, maintain the pH of thestabilization reaction mixture at about 6-10, about 7-10, or preferablyabout 7.0-7.4. Nonetheless, the stabilization method described hereincan be used even at lower pH values. When the bioprosthesis comprises(or is entirely made from) a synthetic material, the stabilizationreaction can be performed within an even broader pH range, such as at apH of about 2-12. The liquid can, for example, be a buffer, such as10-500 millimolar sodium or potassium HEPES buffer at a pH of about 6.9to 7.9 or a 10-500 millimolar sodium or potassium borate buffer at a pHof about 8.5 to 9.5. The buffer is preferably used in excess, relativeto the amount of polyepoxy amine compound that is present, or evenrelative to #×the amount of polyepoxy amine compound, where # is theaverage number of epoxy moieties per molecule of the polyepoxy aminecompound. As an alternative to using an excess of buffer, a reactionmixture having a lower amount of buffer can be used if the mixture isreplaced with fresh (i.e. non-reacted) reaction mixture from time totime. In this alternative, the pH of the reaction mixture can bemonitored, and the reaction mixture can be replaced or supplemented withadditional buffer upon detection of a significant pH change. The buffershould be selected such that it does not chemically react with thepolyepoxy amine compound or the bioprosthesis in a manner that wouldinhibit reaction of the polyepoxy amine compound with the bioprosthesis.Most common buffers (e.g. borate, HEPES, carbonate, cacodylate, citrate,TRIS, and MOPS) are suitable for use.

[0050] The pH of the stabilization reaction mixture can increase as thereaction between the polyepoxy amine compound and the bioprosthesisproceeds. Preferably, the pH of the reaction mixture is maintained at orbelow a maximum value (e.g. at or below pH 10, preferably at or below7.4). pH maintenance can be achieved by any known method, includingacidification of the mixture, addition of buffer, and replacement of thereaction mixture with fresh reaction mixture having a desirable pH (e.g.pH 7-7.4). For example, the reaction mixture can comprise 100 millimolarHEPES and 100 millimolar TGA, can have a pH of about 7.0, and can bereplaced on a daily basis.

[0051] The duration of the period during which the bioprosthesis and thepolyepoxy amine compound are maintained in contact can vary from about 3hours to several months or longer. The duration of contact is preferablyat least about 3 days, and more preferably, at least about 7 days.Contacting the bioprosthesis and the polyepoxy amine compound in anaqueous liquid for 8-10 days is considered sufficient.

[0052] The temperature of the stabilization mixture can be substantiallyany temperature at which the reaction proceeds at an appreciable rateand at which the bioprosthesis is not damaged. It is understood that therate of reaction increases with increasing temperature. If thebioprosthesis contains protein (e.g. if it comprises a tissue obtainedfrom an animal), then the temperature of the reaction mixture can bemaintained, for example, at 20-37° C.

[0053] Pre-implantation treatment with a polyepoxy amine compound can beused to stabilize bioprostheses such as artificial hearts, heart valveprostheses, vascular grafts, annuloplasty rings, dermal grafts, duramater grafts, pericardium grafts, cartilage grafts or implants,pericardium grafts, ligament prostheses, tendon prostheses, urinarybladder prostheses, pledgets, sutures, permanently in-dwellingpercutaneous devices, surgical patches, vascular, cardiovascular, orstructural stents, coated stents and catheters, vascular orcardiovascular, shunts, and the like. Preferably, the bioprostheticdevice is a heart valve prosthesis. Biological tissue treated with apolyepoxy amine compound prior to implantation can be obtained from therecipient, from an animal of the same species as the recipient, or froman animal of a different species than the recipient. Exemplary donoranimals include mammals such as humans, cows, pigs, dogs, and seals, andkangaroos. Exemplary tissues include hearts, heart valves, aortic roots,aortic wall, aortic leaflets, pericardial tissues (e.g. pericardialpatches), connective tissues, dura mater, bypass grafts, tendons,ligaments, dermal tissues (e.g. skin), blood vessels, umbilical tissues,bone tissues, fasciae, and submucosal tissues. The tissue can,alternatively, be a cultured tissue, a prosthesis containingextracellular matrix obtained from an animal, a reconstituted tissue(e.g. bone cells in an artificial bone-like medium), or the like. Thestabilization method described herein can also be used to stabilizebioprostheses comprising one or more materials of non-biological origin,wherein the material has surface chemical groups like those of abiological tissue (e.g. as with a protein-containing synthetic matrix).

[0054] Synthetic analogs of bioprosthetic tissues can be formed fromsynthetic polymers, biological polymers, or both, including thosegenerally found in natural tissue matrices. Suitable synthetic polymersinclude, for example, polyamides and polysulfones. Biological polymerscan be naturally-occurring or produced in vitro by, for example,fermentation and the like. Purified biological polymers can beappropriately formed into a substrate by techniques such as weaving,knitting, casting, molding, extrusion, cellular alignment, and magneticalignment. Suitable biological polymers include, without limitation,collagen, elastin, silk, keratin, gelatin, polyamino acids,polysaccharides (e.g. cellulose and starch), and copolymers of any ofthese. For example, collagen and elastin polymers can be formed into asynthetic bioprosthetic tissue analog by any of a variety of techniques,such as weaving and molding. Synthetic tissue analogs mimic a naturaltissue matrix. Alternatively, synthetic substrates can be used to form atissue analog, either alone or together with naturally-occurringsubstrates. Synthetic tissue analogs can be implanted with or withoutcells seeded on or within them. Such tissue analogs can, optionally, beresorbable.

[0055] The bioprostheses, compositions, and methods described herein arenot limited to those which include a polyepoxy amine compound as thesole bioprosthesis-reactive agent. Other agents, such as additionalcross-linking reagents, calcification inhibitors, GAG-stabilizingagents, and the like can be used in conjunction with (i.e. before,during, or after) polyepoxy amine treatment.

[0056] Cross-linking reagents known in the art include glutaraldehyde,other dialdehydes, carbodiimides, polyepoxy ethers, and the like.Glutaraldehyde is an effective and widely used cross-linker, but, asdiscussed above, its use can lead to unacceptable levels ofcalcification in a bioprosthesis. Other cross-linking reagents which donot necessarily react with the bioprosthesis itself include, forexample, chain extenders and dicarboxylic acids. A polyepoxy aminecompound can be used alone to provide adequate cross-linking. However, agreater degree of cross-linking can further enhance the mechanicalstability of the prosthesis. Therefore, it can be advantageous to useother cross-linking reagents in conjunction with a polyepoxy aminecompound in order to obtain a higher degree of cross-linking in thebioprosthesis. Accordingly, the present invention includes using apolyepoxy amine compound alone or in combination with one or more othercross-linking reagents.

[0057] Use of epoxide-containing compounds, but not polyepoxy amines,for the pre-implantation treatment of biological tissue is known in theart. One such compound is Denacol 521™ (Nagase Chemical Co., Japan),which is a mixture of oligomeric polyglycerol polyglycidyl ethers havingthe general formula I, wherein the most abundant components of themixture have n=2 and n=3.

[0058] Denacol 521™ acts as a cross-linking reagent, and, like otherprior art epoxide-containing compounds, must be used in combination withan alcohol and a catalyst in order to achieve adequate cross-linking ofa biological tissue. In contrast to these reagents, a polyepoxy aminecompound can be used to stabilize a bioprosthesis in the absence ofalcohol and without additional catalysts. It is believed that theincreased efficiency of the cross-linking associated with the use of apolyepoxy amine compound relative to cross-linking associated with useof prior art epoxide compounds is attributable, at least in part, to theenhanced reactivity of the polyepoxy amine, relative to the reactivityof epoxide compounds which do not contain an amine group. As a result, apolyepoxy amine compound, such as TGA, readily undergoes reactions withchemical groups common to proteins such as thiol, hydroxyl, amine, andcarbonyl groups, and does not require the addition of alcohol or acatalyst. Furthermore, as described above, polyepoxy amine compoundscontain an epoxy-reactive moiety (i.e. the amine moiety), so thesecompounds can form covalent linkages with both a bioprosthesis and witheach other.

[0059] In addition to covalently linking chemical moieties of abioprosthesis, treatment of a bioprosthesis with a polyepoxy aminecompound inhibits post-implantation calcification associated with theprosthesis. Examples of prior art calcification inhibitors includeethanol, aluminum chloride, chondroitin sulfate, propylene glycol, alphaamino oleic acid, surfactants, detergents, and the like. In contrast tothese compounds, which require additional reagents to substantiallyenhance the mechanical durability of bioprostheses with which they arecontacted, polyepoxy amine compounds, unaccompanied by additionalreagents, can both covalently link chemical moieties and substantiallyimprove calcification resistance in the bioprosthesis.

[0060] Stabilization of a bioprosthetic tissue can be enhanced bystabilizing GAGs which occur endogenously in the tissue. Examples ofGAG-stabilizing reagents include carbodiimides such as1-ethyl-3-(3dimethyl-aminopropyl) carbodiinide (EDAC), heterofunctionalazides, and carbohydrate-protein linking reagents. Combined use of aGAG-stabilizing reagent and a polyepoxy amine compound to treat animplantable bioprosthetic device can enhance stabilization of thebioprosthesis relative to treatment with the polyepoxy amine compoundalone. Therefore, the invention encompasses using a polyepoxy aminecompound and a GAG-stabilizing reagent, either simultaneously orsequentially in either order, for stabilization of a bioprosthetictissue. The GAG which is stabilized in and on the bioprosthesis can beeither endogenous or exogenous.

[0061] The invention includes a bioprosthesis stabilized using apolyepoxy amine compound according to the stabilization method describedherein. For example, a biological tissue can be harvested from an animalsource (e.g. by removing aortic leaflets from a pig or by obtaining aheart valve from a cow), and processed in vitro using a polyepoxy aminecompound, as described herein. Following this stabilizing treatment, thestabilized tissue can be implanted into a recipient (e.g. a human inneed of a replacement heart valve). Tissue harvesting and implantationmethods are well known in the art; substantially any such method ormethods can be used in conjunction with the bioprosthetic stabilizationmethod described herein.

[0062] Harvested animal tissue can be manipulated (e.g. by combining itwith a material of non-biological origin, such as a polymeric stent orsupport) in vitro, either before or after stabilization of the tissue,prior to implanting the tissue. The tissue can, of course, also betreated using one or more additional reagents (e.g. GAG-stabilizingreagents or calcification inhibitors) prior to implanting it.

[0063] Synthetic analogs of a bioprosthetic tissue such as tissueengineered constructs and artificial cells, tissues, or organs, whichare composed of biological components, synthetic components, or acombination of both, and which have been stabilized using a polyepoxyamine compound are encompassed by the present invention. Examples ofstabilized synthetic prostheses include microencapsulated cells ortissues, and protein-coated prostheses such as catheters, stents, andartificial joints.

[0064] Bioprosthetic tissues, and synthetic analogs thereof, encompassedby the present invention include those which, as a result ofstabilization using a method disclosed herein, demonstrate one or moreimproved or more natural mechanical properties following implantation ina recipient, relative to those stabilized using prior art methods. Suchproperties include, but are not limited to, strength, flexibility,(lowered or negligible) toxicity, and compatibility with normal cellin-growth by the surrounding tissue of the animal. Bioprosthetic tissuetreated as described herein thus more closely resembles native tissue inappearance. For example, soft tissues treated as described herein arenaturally pliable to the touch, rather than stiff. Increased flexibilityimproves the mechanical performance of the tissue followingimplantation, when compared to glutaraldehyde-fixed tissue.Stabilization of tissue using polyepoxy amine compounds also yieldsbetter hemodynamics, improved functionality, and improvedbiocompatibility.

[0065] The invention further includes compositions comprising at least apolyepoxy amine compound and, optionally, one or more of a bufferingagent, a physiological salt (e.g. NaCl, KCl), glycosaminoglycan, aglycosaminoglycan-stabilizing reagent, a second cross-linking agent, anda calcification inhibitor. These compositions can further compriseaqueous and non-aqueous liquids, blood or blood products, and otherliquids which facilitate processing of a bioprosthesis using a polyepoxyamine compound as described herein. Compositions prepared and packagedspecifically to use for stabilizing a bioprosthesis, such as powdered,mixed components of a composition, and frozen or concentrated componentsof a composition, are also included in the present invention.

[0066] The invention is now described with reference to the followingExample. This Example is provided for the purpose of illustration only,and the invention is not limited to this Example, but rather encompassesall variations which are evident as a result of the teaching herein.

EXAMPLE

[0067] The experiments presented in this Example illustrate use oftriglycidyl amine (TGA) to stabilize a bioprosthetic tissue and improveits resistance to calcification.

[0068] The materials and methods used in the experiments presented inthis Example are now described.

[0069] Synthesis of TGA

[0070] TGA (compound III in FIG. 1) was prepared as follows. A 24%solution of ammonia (27 milliliters aqueous NH₃, 0.38 moles) was addedto a solution comprising an excess (120 milliliters, 1.53 moles) ofepichlorohydrin (compound I in FIG. 1), 810 milligrams (4.85 millimoles)of ammonium triflate, and 150 milliliters of isopropanol (I-PrOH). Theresulting mixture was maintained at 20-25° C. for 25 hours withintermittent cooling in a water bath. After adding an additional 30milliliters (0.38 moles) of epichlorohydrin, the mixture was maintainedfor an additional 25 hours at 20-25° C. The resulting solution wasdiluted by adding 120 milliliters of isopropanol, and then concentratedunder reduced pressure at 20-30° C. to yield a viscous syrup. This syrupwas maintained at 30° C. for an additional 2.5 hours. The residue leftafter this procedure was dissolved in 150 milliliters of toluene andconcentrated under reduced pressure at 30-40° C. to yieldtris-(3-chloro-2-hydroxypropyl)amine (compound II in FIG. 1). CompoundII was dissolved in a mixture of 210 milliliters of toluene and 25milliliters of tetrahydrofuran.

[0071] A solution comprising 136 grams (3.4 moles) of sodium hydroxidedissolved in 136 milliliters of water was added to the solution ofcompound II over a period of 0.5 hours at 18-22° C. This reactionmixture was vigorously stirred with intermittent cooling in an ice bath,maintained at 18-22° C. for an additional 2 hours, and then diluted with272 milliliters of water. The temperature of the mixture was not allowedto exceed 30° C. during the dilution. The organic layer was separatedfrom the aqueous layer and dried overnight in the presence of anhydrouspotassium carbonate at 5° C. After removing the desiccant by filtration,the solution was concentrated under reduced pressure, and the residuewas thereafter distilled in a vacuum apparatus at 1 millimeter ofmercury using a 50 centimeter Vigreux fractionating column to yield 42.0grams of TGA (a 65% theoretical yield relative to ammonia). TGA wasrecovered as a viscous liquid, having a boiling point of 98-101° C.Liquid TGA solidified upon refrigeration and remained a solid whenreturned to room temperature.

[0072] TGA, synthesized as described above, was verified by ¹H NMRspectroscopic analysis. The ¹H NMR spectrum of TGA in CDCl₃ is shown inFIG. 2 and indicates that the TGA prepared by this method is a mixtureof two diastereomers, which are present in a ratio of approximatelythree to one. The more abundant diastereomer is thought to be a racemicmixture of the R,R,S and R,S,S enantiomers, and the less abundantdiastereomer is thought to be a mixture of the R,R,R and S,S,Senantiomers. The chemical shifts of the ¹H NMR spectrum in FIG. 2 aredescribed separately here for each diastereomer. For the more abundantisomer, the following NMR data were obtained: δ, ppm, 2.54 (m, 6H, epoxyCH₂), 2.78 (m, 3H, epoxy CH), 3.13 (m, 6H, NCH₂). For the less abundantisomer, the following NMR data were obtained: δ, ppm: 2.26 (dd, 14 Hzand 7 Hz, 3H, H_(A) of epoxy CH₂), 2.72 (dd, 14 Hz and 6.5 Hz, 3H, H_(B)of epoxy CH₂), 2.78 (m, 3H, epoxy CH), 3.04 (dd, 14 Hz and 3 Hz, 3H,H_(A) of NCH₂), 3.29 (dd, 14 Hz and 2.5 Hz, 3H, H_(B) of NCH₂). Adiastereomeric composition is also evident in the ¹³C NMR spectrum ofTGA reported in the literature (Everett et al, 1976, Org. Mag. Res.8:275-276).

[0073] Bioprosthetic Heart Valve Cross-linking Procedures

[0074] In a first method of cross-linking bioprosthetic tissue, fourseparate cross-linking solutions were prepared:

[0075] 1) 0.1 molar TGA in 0.05 molar HEPES buffer at pH 7.4,

[0076] 2) 0.1 molar TGA in 0.05 boric acid-borax buffer at pH 9.0,

[0077] 3) 0.1 molar Denacol 521™ (Nagase Chemical Co., Japan) in 0.05molar HEPES buffer at pH 7.4, and

[0078] 4) 0.05 molar HEPES buffer (pH 7.4) containing 0.6% (v/v)glutaraldehyde.

[0079] Twenty fresh porcine aortic cusps were added to separate 100milliliter aliquots of each of the four solutions (i.e. using a total ofeighty cusps). The cusps and solutions were maintained with constantshaking at room temperature for up to ten days without changing thesolution. At selected times, a small section was removed from each ofthe cusps in each of the cross-linking reactions and analyzed bydifferential scanning calorimetry to determine the thermal shrinkagetemperature (Ts). Cross-linking reactions were considered complete whenthe value of Ts remained substantially constant over time. Cusps treatedin this manner were maintained in the cross-linking solution for morethan 20 days prior to use in subsequent experiments.

[0080] In a second method of cross-linking bioprosthetic tissue,separate 100 milliliter aliquots of 0.1 molar TGA in 0.10 molar HEPESbuffer at pH 7.4 were prepared. The following were added to individualaliquots of this solution.

[0081] i) twenty fresh porcine aortic cusps,

[0082] ii) an amount of bovine pericardium comparable by weight to theamount of cusp tissue, and

[0083] iii) porcine aortic wall segments in an amount comparable byweight to the amount of cusp tissue.

[0084] The tissues and solutions were maintained at room temperaturewith constant shaking for up to seven days, during which time the pH ofeach cross-linking reaction was monitored. For selected reactions, thereaction mixture solution was replaced every 24 hours with afreshly-prepared 100 milliliter aliquot of 0.1 molar TGA in 0.10 molarHEPES buffer at pH 7.4. At selected times, a small section was removedfrom each tissue in each of the stabilization reaction mixtures, and thesection was analyzed by differential scanning calorimetry to determinethe thermal shrinkage temperature (Ts). After seven days of reaction,one group of each of the tissues was transferred to 100 milliliters of0.05 molar phosphate buffer containing 4% (v/v) formalin and stored inthis solution prior to use in subsequent experiments.

[0085] Subdermal Calcification Studies

[0086] Subdermal calcification studies were performed in rats usingtissues treated and stored as described above. Following treatment byeither of the two methods described above, the tissues were rinsedthoroughly with excess sterile saline solution immediately prior toimplantation. Tissues were implanted subdermally (1 tissue sample perrat, 10 rats per treatment method). Twenty-one days followingimplantation, the tissues were recovered and their calcium content wasmeasured in samples taken from each tissue.

[0087] The results of the experiments presented in this Example are nowdescribed.

[0088] pH Monitoring of Cross-Linking Reactions

[0089] As indicated in FIG. 3, the pH of the stabilization reactionmixture using the first stabilization method described herein increasedduring the seven-day reaction period to nearly 11.5. In contrast, whenthe reaction mixture solution was replaced every 24 hours during thereaction, as described in the second method described herein, the pH didnot increase above about 8. Because extremes in pH are not beneficialfor preparation of bioprosthetic materials, maintenance of the pH nearerphysiological pH (i.e. 7.4) during the stabilization reaction is asignificant advantage of the second method, relative to the firstmethod.

[0090] Relative Biocompatibility of Glutaraldehyde-Treated andTGA-Treated Collagen Surfaces

[0091] Denatured (100° C., 1 hour) bovine type I collagen was applied asa film to duplicate culture surfaces. One surface was treated bycontacting it with a solution comprising 0.2% (v/v) glutaraldehyde at pH7.4 for 24 hours at room temperature (i.e. about 20° C.). Anothersurface was treated by contacting it with a solution comprising 0.1molar TGA at pH 7.4 for 24 hours at 37° C. Both surfaces were rinsedextensively using phosphate-buffered saline (pH 7.4) in order to removenon-reacted compounds.—A10 cells (obtained from the American TypeCulture Collection; Gaithersburg, Md.) were provided to both surfaces,and survival of the cells was monitored. A high proportion (estimated at30% or more of cells) which were provided to the glutaraldehyde-treatedcollagen surface appeared to be dead following about 24 hours ofculturing, with significant amounts of cell debris observed. A muchlower proportion (i.e. a substantially undetectable proportion) of cellswhich were provided to the TGA-treated collagen surface appeared to bedead following about 24 hours of culturing. These results demonstratethat TGA treatment of a proteinaceous surface yields a morebiocompatible and less cytotoxic surface than treatment of the samesurface with glutaraldehyde.

[0092] Bioprosthetic Heart Valve Cross-linking

[0093] As indicated in FIG. 4, cross-linking of porcine aortic cuspsoccurs substantially more rapidly using glutaraldehyde than it doesusing TGA. Slower cross-linking kinetics using TGA is beneficial forpre-implantation treatment of bioprostheses because it allows a moregradual cross-linking of structural proteins, thereby yielding aprosthesis that is more durable, more flexible, less toxic, and morecompatible with cell in-growth than glutaraldehyde-fixed prostheses. Theresults shown in FIG. 4 indicate that the final degree of proteincross-linking in tissues treated using TGA can be less than the degreeachieved using glutaraldehyde, at least after ten days of TGA treatment.For this reason, it can be beneficial to use an additional cross-linkingagent as well.

[0094] The data presented in FIG. 5 indicate that the final degree ofprotein cross-linking for cusp tissue treated using a solutioncomprising 0.1 molar TGA and 0.1 molar HEPES, wherein the solution wasreplenished every 24 hours, was lower after seven days of the reactionthan the degree of cross-linking obtained by glutaraldehyde treatmentafter seven days. However, the degree of cross-linking in thepH-controlled reaction was substantially higher than the degree ofcross-linking obtained when the same TGA solution was used withoutreplenishing the solution during the reaction. Furthermore, TGAtreatment in which the solution was replenished yielded cusp tissuehaving a Ts of about 83° C., which is comparable to glutaraldehyde-fixedtissue. Thus, controlling the pH of the reaction mixture can mitigatethe need to use an additional cross-linking agent.

[0095] Subdermal Calcification Studies

[0096] The calcium content of individual tissue samples (in microgramsof calcium per milligram of tissue) treated using the two methodsdescribed in this Example are listed in Table 1 and Table 2.

[0097] Values in Table 1 were obtained using cusp tissue treated usingthe first stabilization method described above. Values in column Acorrespond to porcine aortic cusps cross-linked using glutaraldehyde.Values in columns B and C correspond to porcine aortic cuspscross-linked with two different batches of Denacol 521™. Values incolumns D and E correspond to porcine aortic cusps cross-linked usingTGA in HEPES buffer and borate buffer, respectively. The values listedat the bottom of each column represent the average value±the standarderror corresponding to the data in that column. TABLE 1 A B C D E 158.700.65 49.92 1.107 1.318 140.34 0.74 90.39 1.021 0.811 141.91 0.81 54.090.615 1.31 94.44 0.52 33.83 1.30 2.13 89.60 0.51 93.59 8.75 1.20 87.420.66 65.01 1.14 3.85 63.28 0.54 68.65 1.59 72.44 0.47 30.15 17.58 117.670.80 66.88 131.43 2.53 63.42 106.69 ± 0.824 ± 0.194 61.30 ± 7.33 4.13 ±2.14 1.769 ± 0.95 9.81

[0098] Values in Table 2 were obtained using tissues treated using thesecond method described above. Values in Group I correspond to tissuescross-linked using glutaraldehyde. Values in Group II correspond totissues cross-linked using the second method described above. Values inGroup III correspond to tissues cross-linked using the secondstabilization method described above, wherein the tissues weresubsequently treated with and stored in a formalin-containing solution.The values listed represent the average value±the standard errorcorresponding to the data in that row. TABLE 2 Calcium (micrograms/Group Tissue milligram of dry tissue) I porcine aortic cusps 126.12 ±8.51  porcine aortic wall 56.35 ± 6.14 bovine pericardium 121.16 ± 7.49 II porcine aortic cusps  2.14 ± 0.69 porcine aortic wall 18.67 ± 1.36bovine pericardium  1.80 ± 0.28 III porcine aortic cusps 18.07 ± 9.19porcine aortic wall 21.17 ± 1.81 bovine pericardium  7.43 ± 3.47

[0099] The results of these experiments indicate that treatment oftissues using TGA, as described above for the first and secondstabilization methods, inhibits post-implantation calcification of thetissue relative to glutaraldehyde treatment of these tissues, and is atleast about as effective at preventing calcification of implanted tissueas polyepoxides such as Denacol 521™. These experiments also indicatethat TGA treatment of aortic cusps does not exhibit the variabilityobserved with Denacol 521™. Furthermore, these experiments demonstratethat controlling the pH of TGA-mediated stabilization reaction mixturesimproves calcification resistance and the degree of cross-linking.

[0100] The experiments presented in this Example demonstrate that thestabilization method described herein can be used to cross-link abioprosthetic tissue prior to implantation to enhance the biomechanicalstability and calcification resistance of the tissue.

[0101] The disclosures of every patent, patent application, andpublication cited herein are incorporated herein by reference.

[0102] While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims include all such embodiments and equivalent variations.

What is claimed is:
 1. An implantable bioprosthesis comprising proteinscross-linked with a poly-(2-hydroxyorgano)amino moiety.
 2. Theimplantable bioprosthesis of claim 1, wherein a methylthio group of thebioprosthesis is linked with the poly-(2-hydroxyorgano)amino moiety. 3.The implantable bioprosthesis of claim 1, wherein an amine group of thebioprosthesis is linked with the poly-(2-hydroxyorgano)amino moiety. 4.The implantable bioprosthesis of claim 1, wherein a phenolic hydroxylgroup of the bioprosthesis is linked with thepoly-(2-hydroxyorgano)amino moiety.
 5. The implantable bioprosthesis ofclaim 1, wherein a phosphate group of the bioprosthesis is linked withthe poly-(2-hydroxyorgano)amino moiety.
 6. The implantable bioprosthesisof claim 1, wherein a carboxyl group of the bioprosthesis is linked withthe poly-(2-hydroxyorgano)amino moiety.
 7. The implantable bioprosthesisof claim 1, wherein substantially all epoxy-reactive groups of thebioprosthesis are linked with the poly-(2-hydroxyorgano)amino moiety. 8.The implantable bioprosthesis of claim 1, wherein the implantablebioprosthesis is selected from the group consisting of an artificialheart, a heart valve prosthesis, an annuloplasty ring, a dermal graft, avascular graft, a vascular stent, a structural stent, a vascular shunt,a cardiovascular shunt, a dura mater graft, a cartilage graft, acartilage implant, a pericardium graft, a ligament prosthesis, a tendonprosthesis, a urinary bladder prosthesis, a pledget, a suture, apermanently in-dwelling percutaneous device, a surgical patch, avascular stent, a cardiovascular stent, a structural stent, a coatedstent, a vascular shunt, a cardiovascular shunt, and a coated catheter.9. The implantable bioprosthesis of claim 8, wherein the implantablebioprosthesis is a heart valve prosthesis.
 10. The implantablebioprosthesis of claim 1, wherein the implantable bioprosthesiscomprises a biological tissue.
 11. The implantable bioprosthesis ofclaim 10, wherein the tissue is selected from the group consisting of aheart, a heart valve, an aortic root, an aortic wall, an aortic leaflet,a pericardial tissue, a connective tissue, dura mater, a bypass graft, atendon, a ligament, a dermal tissue, a blood vessel, an umbilicaltissue, a bone tissue, a fascia, and a submucosal tissue.
 12. Theimplantable bioprosthesis of claim 10, wherein the tissue is harvestedfrom an animal.
 13. The implantable bioprosthesis of claim 12, whereinthe animal is selected from the group consisting of a human, a cow, apig, a dog, a seal, and a kangaroo.
 14. The implantable bioprosthesis ofclaim 1, wherein the implantable bioprosthesis comprises a syntheticanalog of a bioprosthetic tissue.
 15. The implantable bioprosthesis ofclaim 1, wherein the poly-(2-hydroxyorgano)amino moiety is apoly-(2-hydroxypropyl)amino moiety.
 16. An implantable bioprosthesiscomprising proteins cross-linked by contacting the bioprosthesis with apolyepoxy amine compound
 17. The implantable bioprosthesis of claim 16,wherein the bioprosthesis is contacted with the polyepoxy amine compoundin an aqueous liquid having a pH of about 6 to
 10. 18. The implantablebioprosthesis of claim 16, wherein the proteins are cross-linked bycontacting the bioprosthesis with the polyepoxy amine compound in anaqueous liquid having a pH of about 7 to
 10. 19. The implantablebioprosthesis of claim 16, wherein the proteins are cross-linked bycontacting the bioprosthesis with the polyepoxy amine compound in anaqueous liquid having a pH of about 7.0 to 7.4.
 20. The implantablebioprosthesis of claim 16, wherein the polyepoxy amine compound istriglycidyl amine.
 21. The implantable bioprosthesis of claim 16,wherein the proteins are cross-linked by contacting the polyepoxy aminecompound with moieties of the proteins which are independently selectedfrom the group consisting of a methylthio moiety, a primary aminemoiety, a phenolic hydroxyl moiety, a phosphate moiety, and a carboxylmoiety.
 22. The implantable bioprosthesis of claim 16, wherein theimplantable bioprosthesis is treated with a second stabilizationreagent.
 23. The implantable bioprosthesis of claim 22, wherein thesecond stabilization reagent is a cross-linking reagent.
 24. Theimplantable bioprosthesis of claim 22, wherein the second stabilizationreagent is a calcification inhibitor.
 25. The implantable bioprosthesisof claim 24, wherein the calcification inhibitor is aluminum chloride.26. The implantable bioprosthesis of claim 22, wherein the secondstabilization reagent is a glycosaminoglycan-stabilizing reagent. 27.The implantable bioprosthesis of claim 26, wherein theglycosaminoglycan-stabilizing reagent is a carbodiimide.
 28. A method ofstabilizing an implantable bioprosthesis, the method comprisingcontacting the bioprosthesis and a polyepoxy amine compound, whereby thebioprosthesis is stabilized.
 29. A composition for stabilizing animplantable bioprosthesis, the composition comprising a polyepoxy aminecompound and at least one of a calcification inhibitor, aglycosaminoglycan-stabilizing reagent, and a second cross-linkingreagent.